This invention relates to a laser scanning microscope that senses the transmitted light or reflected light from a specimen or fluorescence from a specimen while scanning the specimen by means of the coherent light emitted from a laser light source.
FIG. 23 shows the configuration of a scanning-type laser microscope.
The scanning-type laser microscope comprises a laser light source 100 that outputs coherent light, a scanning optical unit 102 that causes a bundle of rays from the laser light source to scan the image plane of an objective 107, and the objective 107 that gathers the bundle of rays of light on the image plane.
The operation of the scanning-type laser microscope will be explained.
The coherent light (laser light) from the laser light source 100 passes through a dichroic mirror 101 and enters the scanning optical unit 102.
The scanning optical unit 102 includes scanning mirrors 102a, 102b that scan at right angles with each other. The scanning optical unit 102 deflects the incident coherent light in the direction of X and the direction Y by means of the scanning mirrors 102a, 102b.
After the deflected coherent light has passed through a relay lens 103, the direction of its optical path is changed by a mirror 104. Then, the coherent light passes through a dichroic mirror 105 and is caused by an image formation lens 106 to meet the pupil diameter of an objective 107.
Specifically, the coherent light passed through the image formation lens 106 reaches a dichroic mirror 108 and an objective 107. The coherent light further passes through the objective 107 and is gathered on the cross section 111 of a specimen 110 placed on a stage 109.
When the coherent light is projected on the specimen 110 this way, the light excites a fluorescent indicator, which then generates fluorescence. For example, when the calcium ion indicator fluo-3 is used as a fluorescent indicator and a laser wavelength of 488 nm (e.g., argon laser) is used, fluo-3 will generate light with a fluorescence wavelength of 530 nm.
The fluorescence from the specimen 110 travels backward on the optical path. Specifically, the fluorescence from the specimen 110 passes through the objective 107, dichroic mirror 108, image formation lens 106, dichroic mirror 105, mirror 104, relay lens 103, and individual scanning mirrors 102b, 102a and reaches the dichroic mirror 101. The dichroic mirror 101 reflects the light, which then enters a photometric filter 112.
The photometric filter 112 permits only the fluorescence wavelength from the specimen 110 to pass through. The light is caused by the lens 113 to form an image in the plane of a pin hole 114. The photometric filter allows a specific wavelength band to pass through. The fluorescence passed through the pin hole 114 is measured by a photoelectric conversion element 115.
The characteristic of the dichroic mirror 101 is determined by the excitation wavelength (laser wavelength) of a fluorescent pigment that dyes the specimen 110 and the fluorescence wavelength. For example, when the calcium ion indicator fluo-3 is used as a fluorescent indicator and a laser wavelength of 488 nm (e.g., argon laser) is used, fluo-3 will generate light with a fluorescence wavelength of 530 nm as described above. This will cause the dichroic mirror 101 to reflect rays of-light with a wavelength of 505 nm or more.
In the observation of the specimen 110, the coherent light is deflected in the direction of X and the direction of Y by the scanning mirrors 102a, 102b in the scanning optical unit 102, passes through the objective 107, and illuminates the specimen 110. This makes it possible to continuously measure the fluorescence in the image formation position on the cross section 111 of the specimen 110 and form an image of the specimen in the scanning range.
The raising and lowering of the stage 109 or objective 107 produces a cross-sectional image different from the cross section 111 of the specimen 110, which makes it possible to form a three-dimensional image of the specimen.
When an UV (ultraviolet rays) pulse laser is used as the laser light source 100 and the scanning mirrors 102a, 102b in the scanning optical unit 102 are stopped in given directions, the UV pulse laser light can be projected on the desired position on the specimen 110.
For instance, when a caged indicator is used, the projection of the UV pulse laser causes the substance enclosed by the caged compounds to be emitted, which induces a peculiar phenomenon in a particular region of the cell.
The entire illumination of the specimen 110 is carried out by an illumination optical system composed of a light source 116, a lens 117, an excitation filter 118, and a dichroic mirror 108. The fluorescence emitted from the specimen 110 is reflected by the dichroic mirror 105. A photometric filter 119 extracts the fluorescence wavelength. Then, a photographing element 120, such as a CCD camera, receives the fluorescence wavelength extracted by the photometric filter 119 and produces an image of the fluorescence.
As described above, when laser light (i.e., UV pulse laser) is projected momentarily at the desired position on the specimen 110 and the dynamic characteristic of the specimen 110 resulting from the effect of the projection of the laser light is determined, a chronological record of the specimen images is needed. To meet the need, a method of acquiring images by use of a CCD camera is generally used.
The method of acquiring images with a CCD camera, however, produces no confocal image. The acquired image has a focal depth greater than that of a confocal image. As a result, it is not known at what depth a peculiar phenomenon resulting from the release of the caged radicals has been developing.
Therefore, it is desirable to determine the dynamic characteristic of the specimen 110 by momentarily projecting laser light at the desired position on the specimen 110 while observing and recording the image of the specimen with a confocal laser microscope.
Depending on uses for research work, the region on which laser light is projected and the cross section the researcher wants to observe are not necessarily in the same plane. There may be a case where the researcher wants to project laser light on part of a cross section and get an image of a different cross section.
For instance, when stimulation is given to the sympathetic nerve outside an artery, it is determined what response has occurred in the smooth muscle or endothelial cells inside the artery.
In such a case, it is desirable that the region on which laser light is projected and the cross section whose image is to be acquired should be selected within the specimen 110.